Method for Creating New Germplasm of Male Sterile Crop by Gene Editing and Application Thereof

ABSTRACT

A method for creating a new germplasm of a male sterile crop by gene editing and an application thereof are provided. In this method, the gene editing is performed on an exon region of a Ty-5 gene, and a deletion of DNA sequence is introduced by using a repair mechanism of plants themselves to double-strand breaks (DSBs), causing a loss of function of Ty-5 gene, thereby obtaining a male-sterile character. The method can be applied without being limited by crop categories. After the gene editing is performed on Ty-5 genes of various crops, new germplasms can be quickly obtained. The new germplasms have the same agronomic characters as the previous materials, and only differ in sexual aspect, which effectively solves the problem of the lack of male sterile materials and unstable fertility in natural resources.

CROSS REFERENCE TO THE RELATED APPLICATIONS

This application is the national phase entry of InternationalApplication No. PCT/CN2018/106586, filed on Sep. 20, 2018, which isbased upon and claims priority to Chinese Patent Application No.CN201810735328.6, filed on Jul. 6, 2018, the entire contents of whichare incorporated herein by reference.

TECHNICAL FIELD

The present invention belongs to the technical field of gene editingtechnology for crop breeding, and particularly relates to a method forcreating a new germplasm of a male sterile crop by a gene editing and anapplication thereof.

BACKGROUND

Crop male sterility is a phenomenon of normal pollination failure due toinability to produce pollen or abort pollen, which is caused by abnormalmale organs resulting from physiological or genetic reasons of sexuallypropagated crops. Because crop male sterility can avoid the artificialemasculation in the pollination process in crop heterosis breeding, alot of labor input is saved. Meanwhile, crop male sterilitysignificantly improves the purity of hybrid varieties, and createsvarieties with heterosis.

Male sterility technology plays an important role in the utilization ofheterosis, and obtaining stable male sterility materials has importantapplication value. Male sterility can be divided into nucleus malesterility and nucleus-cytoplasmic male sterility according to a geneticmodel and position of male-sterile character in cells. At present, malesterility has been applied in crops such as rice, corn, wheat, cabbage,pepper and the like. However, in many crops, male sterility is not usedfor production mainly due to problems like lack of male sterileresources and stability of male-sterile character of male sterilematerials. It is found that the male sterile line material with naturalmutation is the main source of male sterile materials. In addition, malesterile materials can be obtained by distant hybridization, artificialmutagenesis, and cell engineering. With the development ofbiotechnology, it has become possible to create male sterile materialsthrough genetic engineering.

The gene Ty-5 is a tomato yellow leaf curl virus resistance gene intomato, and is also a surveillance factor for monitoring a peptide chainsynthesis process. The gene Ty-5 exists in all crops. In the presentapplication, the new germplasm of male sterility can be created rapidlyby performing a gene editing on the gene Ty-5. The development of geneediting technology provides a powerful weapon for the utilization ofTy-5 gene in heterosis breeding. The current gene editing technologiesmainly include zinc finger nuclease technology, transcriptionactivator-like effector nuclease technology, and the latest CRISPR/CAS9gene editing technology. Gene editing realizes the recognition andcleavage of specific DNA sequences, and the introduction of differenttypes of mutations such as deletion, substitution, and insertion ofbases at double-strand breaks (DSBs) of DNA, achieving fixed-pointediting of DNA.

SUMMARY

In view of the problems of the shortage of male sterile materials andthe low purity of hybrid varieties, and the current situation of longtime and high cost of conventional breeding, gene editing technology isapplied to Ty-5 gene according to the present invention, so as torapidly create a new germplasm of a male sterile crop while retainingagronomic characters of an original male fertile material.

In order to solve the above technical problems, the following technicalsolutions of the present invention are used.

(1) All of the existing gene editing methods can be used for the editingof Ty-5 in this study. In the present invention, only CRISPR/Cas9technology is used for the gene editing of Ty-5. A deletion of DNAsequence is introduced by using a repair mechanism of plants themselvesto DSBs, causing a loss of function of Ty-5 gene, thereby obtaining amale-sterile character;

(2) gRNA target sites are selected in an exon region of Ty-5 gene.According to the principle of CRISPR/Cas9 target anchor, the 18-20 bpupstream of the protospacer-associated motif (PAM) as the target site,i.e., (5′-N18-20NGG-3′, NGG is a PAM sequence, and N18-20 represents arecognition sequence of 18-20 bp);

(3) Oligo sequence primers are designed based on the recognitionsequence:

Target-Sense: 5′-TTG-NNNNNNNNNNNNNNNNNNN (N represents gRNAsensesequence) Target-Anti: 5′-AAC-NNNNNNNNNNNNNNNNNNN N represents thereverse complement of gRNA sense)

The primers are respectively diluted to a concentration of 10 μM, 5 μLof each of the above primers is taken, and 15 μL of water is added for auniform mixing to form a mixed solution. The mixed solution is placed at95° C. for 3 minutes, then slowly cooled to 25° C., and finally kept at16° C. for 5 minutes to complete a synthesis of an oligo dimer;

(4) A CRISPR/Cas9 kit VK005-14 from VIEWSOLID BIOTECH, Beijing isselected, 1 μL of synthesized oligo dimer, 1 μL of Cas9/gRNA vector, 1μL of Solution 1, 1 μL of Solution 2, and 6 μL of H₂O are mixeduniformly to form a mixed solution, and a reaction is performed on themixed solution at 16° C. for 2 h;

(5) After ligation, the vector is transferred into E. coli competentstate, a single clone is picked for plasmid sequencing analysis. Asequencing primer is sqprimer: 5′-GATGAAGTGGACGGAAGGAAGGAG-3′, and apositive plasmid is transferred to Agrobacterium GV3101;

(6) A cotyledon of the crop is used as an explant, a plant regenerationis carried out by a leaf disc method, Agrobacterium GV3101-mediatedtransformation is performed, and a regenerated plant is obtained byhygromycin resistance screening;

(7) Primers that can amplify the target site are designed according toupstream and downstream sequences of the target site, and theregenerated plant DNA is extracted to be used as a template for PCRamplification. The amplified products are sequenced, and a sequencinganalysis is performed on the regenerated plants to determine whether agene editing occurs and whether an editing type is homozygous mutation;

(8) After the sequencing analysis, a pollen content and germinationidentification is performed on homozygous gene edited strains. Theresults show that the homozygous edited Ty-5 plants created by the geneediting are new male sterile germplasms.

Compared with the prior art, the advantages of the present invention areas follows:

(1) The method for creating a new germplasm of a male sterile crop ofthe present invention can be applied without being limited by cropcategories. After the gene editing is performed on Ty-5 genes of variouscrops, new germplasms can be quickly obtained. The new germplasms havethe same agronomic characters as the previous materials, and only differin sexual aspect, which effectively solves the problem of the lack ofmale sterile materials and unstable fertility in natural resources.

(2) As compared to a conventional breeding method, materials treated bythe method of the present invention can obtain male-sterile charactersmore quickly.

(3) As compared to a conventional breeding method, materials treated bythe method of the present invention can better retain the agronomictraits of the acceptor material.

(4) The new male sterile germplasm created by the present invention is apowerful supplement to existing materials.

(5) Performing a hybrid seed production by using the materials createdby the male sterility creating method of the present invention cangreatly reduce the labor requirement and improve the purity of thehybrid seed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing a regenerated plant after a gene editingamplified by a gene editing primer;

(M: 100 bp Marker; 1: regenerated plant No. 1 after the gene editing; 2:regenerated plant No. 2 after the gene editing; 3: wild type Money makermaterial);

FIG. 2 is a diagram showing results of a sequencing of each materialband amplified by a CRISPR5-F primer;

(a: regenerated plant No. 1; b: a sequencing result of a wild typemoneymaker; c: regenerated plant No. 2; triangle boxes indicate sequencedeletions of the regenerated plant No. 1 and the regenerated plant No.2, respectively); and

FIG. 3 is a diagram showing a comparison of pollen germination;

(a: regenerated plant No. 1; b: wild type moneymaker; c: regeneratedplant No. 2).

DETAILED DESCRIPTION OF THE EMBODIMENTS

The technical solutions of the present invention will be clearly andcompletely described with reference to the drawings in the embodiments.It is obvious that the described embodiments are only a part of theembodiments of the present invention, but not all embodiments. All otherembodiments obtained by those of ordinary skill in the art based on theembodiments of the present invention without creative work should beconsidered as falling within the scope of the present invention.

A method for creating a new germplasm of a male sterile crop through agene editing, includes the following steps:

(1) Materials

Material of moneymaker having normal stamen fertility;

CRISPR/Cas9 Kit: a CRISPR/Cas9 Kit VK005-14 from VIEWSOLID BIOTECH,Beijing was selected;

T carrier: TransGen Biotech, Beijing. (CT301-01);

DNA Extraction Kit: TIANGEN Biotech (Beijing) Co., Ltd. (DP305);

Agrobacterium competent state: GV3101;

Primer synthesis and sequencing were completed by GenScript BiotechCorp. (Nanjing).

(2) Selection of gRNA Target Sites

The full length sequence of the Ty-5 gene (as shown in SEQ ID NO: 1)includes a total of 16 exons, and the second exon sequence was selectedfor the design of the gRNA target site in the present embodiment. Thelength of target sequence is 19 bp (as shown in SEQ ID NO: 2). Theprimers were designed based on the target sequence:

Target-F: 5′-TTG-AGAAGAAGCTGATGATCTA-3′, as shown in SEQ ID NO: 3,Target-R: 5′-AAC-TAGATCATCAGCTTCTTCT-3′, as shown in SEQ ID NO: 4;

(3) Oligo Dimer Synthesis:

The Target primers were respectively diluted to a concentration of 10μM, 5 μL of each of the above primers was taken, and 15 μL of water wasadded for a uniform mixing to obtain a mixed solution. The mixedsolution was placed at 95° C. for 3 minutes, slowly cooled to 25° C.,and then kept at 16° C. for 5 minutes, so as to complete the synthesisof oligo dimer;

(4) Construction of CRISPR/Cas9 Recombinant Vector and Transformation ofAgrobacterium

The reaction system was as follows: 1 μL of synthesized oligo dimer, 1μL of Cas9/gRNA vector, 1 μL of Solution 1, and 1 μL of Solution 2, and6 μL of H₂O were mixed homogeneously to obtain a mixed solution, and themixed solution reacted at 16° C. for 2 h. The ligated vector wastransformed into Escherichia coli competent Trans1-T1, and coated on aplate for placing overnight. A single colony was selected for extractingthe plasmid, and a sequence analysis was performed on the plasmid. Thesequencing primer was sqprimer: 5′-GATGAAGTGGACGGAAGGAAGGAG-3′, as shownin SEQ ID NO: 5. The plasmid was extracted from the colony with thecorrect sequencing result, and transferred into the Agrobacterium GV3101by a freeze-thaw method.

(5) Tomato Transformation:

The main steps were as follows:

Pre-culture preparation of explants: Cotyledons that had just emergedfrom the seed coat were selected for transformation. The cotyledons werecut and placed in 50-100 mL of MS liquid medium. The stalk ends and tipsof the cotyledons were excised and placed in D1 medium, and thecotyledons were placed upside down for culture in D1 medium. The culturedish was pre-incubated for 2 days in a constant temperature culturechamber (16 L/8 D) at 24° C.

Co-culture: 5 μL of 0.074 M acetosyringone was added to each 10 mL of MSliquid medium, 5 mL of the MS liquid medium (+AS) was taken to washAgrobacterium colonies, the bacterial solution OD600 was diluted to aconcentration of 0.3-0.4, and the bacterial solution was added to thepre-cultured (2 days) cotyledons. After infecting the cotyledons for8-10 minutes by Agrobacterium, the excess bacterial solution wasremoved. The cotyledons were transferred to the D1 medium containingfilter paper. The cotyledons were inversely placed (the lower surface ofthe cotyledon faces upwards) for incubation at 24° C. for 2 days in thedark.

Screening: the sterile cotyledons were transferred to a 50 mL centrifugetube, washed twice with sterile water, and washed once with +Car; thecotyledons were inversely placed (back up) on the differentiation medium2Z, 20-30 leaves were placed per culture dish to ensure the growth spacerequired for cotyledons. The double-layer sealing film was used forsealing, and a cultivation was performed at 24° C. for 10 days (16 L/8D). On the tenth day, the cotyledons were transferred to fresh 2Z mediumplates, and then subcultured every 2-3 weeks. The callus appeared after2-3 weeks. The initial buds appeared within 4-6 weeks. When the budsbegan to appear, the explants were subcultured every 2 weeks, and themedium was 1Z selective medium.

Rooting: the shoots were picked from the explants and placed in a 100 mLsterile vial containing 40 mL of MMSV medium (containing 0.5 mg/L IBA,15 mg/L hygromycin and 200 mg/L Timentin). Roots began to appear aftercultured for around 2 weeks. When the plants grown large enough, theseedlings were transplanted into small plastic pots containing a mixtureof vermiculite and soil, and watered with nutrient water. A total of 2transformed tomato seedlings were obtained by tomato transformation.

6. Regenerated Plant Detection after Gene Editing:

Leaf DNA of two regenerated plants and wild type moneymaker materialswere extracted separately, and the following amplification primers wereused:

CRISPR5-F: 5′-TCCATTGAACTGAAGCAAATCTC-3′, as shown in SEQ ID NO: 6;CRISPR5-R: 5′-GCTAATAATGCTAAGCCCTCACA-3′, as shown in SEQ ID NO: 7.

The length of the amplified fragment was about 450 bp, and the productafter PCR amplification was subjected to an agarose gel electrophoresisto determine whether the size of the strip was correct (FIG. 1). Theelectrophoresis bands showed that the regenerated plants and wild typemoneymaker materials could amplify a 450 bp band, and the three PCRproducts amplified by the regenerated plants and wild materials weresequenced by CRISPR5-F primers to detect the variation of the basesequence of the gene editing site (FIG. 2). The results showed that, ascompared to wild type moneymaker materials, using the regenerated plantsNo. 1 and No. 2 resulted in sequence variation at the gene editing site.However, the variation type of the plant No. 1 was heterozygousmutation, and a 13 bp deletion occurred in one of the double-strandedDNA; the variation type of the plant No. 2 was homozygous mutation, anda 6 bp deletion occurred respectively at a same site of thedouble-stranded DNA.

7. Comparison of Pollen Content and Germination

The pollens of two regenerated plants and wild type moneymaker werecollected, and incubated on the germination medium for 4 h at 26° C. inthe dark. The pollen contents and the germination thereof of variousmaterials were compared and observed under a microscope (FIG. 3).Through the germination comparison test, the pollen content ofregenerated plant No. 1 was equal to that of wild type moneymaker, whilethe pollen content of regenerated plant No. 2 was significantly reduced.By the comparison of pollen germination, no pollen germination wasobserved in plant No. 2 after cultured for 4 h, but pollen germinationwas observed respectively in regenerated plant No. 1 and wild typemoneymaker. It can be seen that homozygous variation (plant No. 2) cangreatly reduce plant pollen content and stop pollen germination.

In summary, the present invention provides a method for creating a newgermplasm of a male sterile crop by a gene editing. Through performing agene editing on the Ty-5 gene exon region, the homozygous edited plantwas obtained, which is a new male sterile germplasm.

SEQ ID NO: 1

The full-length sequence of the Ty-5 gene in the tomato materialmoneymaker, the underlined sequence is the intron sequence, and theshadow indicates the gene recognition site.

ATGAAGATTGTTCGTAGAGACTTTGTTCCTGATGGTTCTGGTAGTGTAAAGGTAACTTTTTTATCTCTATAATTGTTGTTTAAATCTATAATTCGAGTAATTTTCGTGATTTTTTGAAACCCCAGATGAAGAAAACGTTAAAATT

TTGCTTATAATCTGATAGCTGAAGGTGATACTGTATTAGCTGTTACTGTTAGGTATTGCACTTTTGCTCAATTTTATTAGTGTGAGGGCTTAGCATTATTAGCAATTTTTTTGGGATAAATAAGTAATTTTTATTCGCGTATGTAATAAGTTTGAGATTTTGTAGAAAAACATGTTTTTACTATAAGAAGCATTTAGTTTATACTACTCCCTTACTCCGTTACAATTTGTTTGTTTGGTTTTGAATTGTCACGAGTTTTTTAAAAAAGAGAGTAAAGAACGACTTTTGAATCTCATGGTCTTTAAACTAAAGAGATTGTGGGATGTACGGAATTTGGTCTTTTATCTTGTGCTATTAAATATGGTAGGTGGAAAGTCGAATAGAAGAGTTGCCAAATAAGGAAAGAGACATTATTTTTGGAACAAACTAAAAAGAAAAGTAGGATAAACAAATTAAAACAGGGGGAGTATTTGGTTTCTCATCGGTGCTACAAGAATTACTAAAAAGCTAGCGTCTTGCCTTTTTTAAAAAGAAATTTTGATCCTGAAAGATGGAGTTTTTAATTTGAATATGGATGTCACTTGCTAATTAGGTTCGAATTCTGTTCATTCGAAAGCTTGGAGCTTTTTACTTTGTTTATAACTACATAGTTGATTCTCTACACTTGTTAATGTTTCTCATCCTGTTGAGTTGATTGAAAATTTATGTTTTTTGGATAACATATTAATTGCTCTCGTTCTCGTAAATGTCTAATTTCTGCATCAATTTTTGTTGTTTTATTCGTTGATTAGCTAATTCTTAGAAAATGTAGTGTTCTGTGATGTGCGAAAGTAATCTATAAATAGTTAGACTCTAATCAGATTTGTGCTAAATTCTAGTTAAATTTGTCTAAATTGGCCTGAGATGAGTTTATATAAACTGTGGAGTTACATGGTCAGTGAGGATTCATTTATACGACCTGAACTTGCTTGGACTGAGGTGTTGTTGTTATTGTTGTTTGCTGAAATTGTGAATGACAATGTTACTATAAGGAAAATGGATTTTGAGCATAGTGTGAGTCTCTACTGATTACACAGGTTGATAATAGTATTGTTATATTTGTTTTGCTTAAAGCTTAGGGGAAGACTCTTTTTTAATGTTGAGAGCAACTTTCTTTTGCAAATTATGCTATGTGGGTCTTTGCTGATTGTTAACACGGTTTAGCTTGTCACAACACAACCTACTGGAAGCTGGTTAAATTTTGCTTGTAATTTCTTGTAGGAAGGTCCTGAGGGAAGCTGCTTCTGGAGGAAGAGATGCTGAACGAGTGAAACTGAAATTGGAAATTAAAGTTGAGGTAAGGATATATTAGACATCCAGCATCATTCAGTTGTGGGGTGCGGGCCTTGGTTAGAACTTTGTTTTAGCTTCCAGCATAAGGTGTTTTAATATAAATTGAAAGATAAAACTTTGAAATCAATTAACTATAGAGAAGAATCTACTAAGGATAGAGAGGAGACTTTTCTTTGTCTTCCTTTTCCAATGTGGTCGAGATGAAGTATTTTTGGGCAGTTTGATGAATTTTGAGGATATCAATACAACCCGTGTATGACATATGCATGATAAATTGTCACATAACAATGCTCCATTTCTAACTAATCTCAATAAATGCGATGTTGATAGTTTTGTTACCATAGTAATGATGACAATTGTAATGTGACTGGAAAGTAGGAAATAGATAGTGCTCTTTCTAGAGTTTTTATTTGAATATATTCCACTCCAATGTGGTATAATATAATCAATTTGGTGTTTTAGAATGTGGAGTATGACAAAGAAGGTTCTGCCTTGCGTATTCGCGGGAAGAATATTCTGGAGAATGAACATGTAAAGGTGTGTATTCTTCAACTTAATCCTTTTGGATAAATTCCTAATATTGATGCTGCAACAAAAATGTATCTAATTATTTTATTGCAGATAGGGGCCTTTCACACTCTGGAAATTGAGCAACACAGACCTTTTGTGCTAAGAAAGGTACAATGCTGTGTTTTGATTCTTTTGCAACTATCACTCTGTTTTCTTTTAAAAATTTTGAGGTATATTATTTGCACTTTAAAAGCTATCAAGCTGGTGATATATGTTCTTTAGGATTGCAGCTCTAACCTATGTTTCTCAGACTCTTCAAAAATGTCAACTGGTGCATGTCGGATTCTCCAAAAATAGCGTGTTTTGGAATATCCGACATGGGTGCGGCATTGTAAGTGAAGAGTCCGCAACTTAGGCTCTAACAAGTAAAAAAATCTGTAAAGTTATTCATGTAATTTACATTATTATTATAAAATCCCCGGAAAAAGAAGAAGAGTACCTTTTTTTTCTTTAATAACCTTGGTATCCGGGCCAGTTTGTGCACACTTCGACCACTTCCACCAACACAGCTACCGCCTACAGGGTAACTCTTTCCATCAAGGTTTTGACAAATAAGAAGAAATTGTCTAGTGTTTTTCGCCTCTGGTTGGATTTGAACCTAAGATCTTATGACTCTTAACTCACTTCATTGGCCGCTAGACCATACCCCTTGGGTGCAGTTACTTGAAACTAAGTTTAATTTTTCCCGCTAAATTTCAGTTGTCCCGCCGCTGACACCCCTTCGAGGCATCTCGTGTTCCTGTCCTTTGACGTAGTTCACATCCTGCACGAAAGGTGTACATACGATACCTCCTTTCTGTCTCTAGATGGAACAACCCTTCTACATTAATGATGATTCCACCTTGGTTATCATCATGACTAATTCCATGCCAAATAAGCCAAGAAATAGCTACACATTTTCGGCACACACCAAAAAAGGGAACCCGAAGTATATTGTCGTTGATTTCTCATAAACAAGTGTACAATGGTTCGAAAATTATCCGAAAGCATTAATGCTTATTAAGCTATTATTAACTCCTATATTATACATTCTATGGACTTTGGTCTGAGGGGGCTAGTTTCTTTTCTCAGCTTGTTTCTTTGTAGTTTGAAGGATCTATGCCCCCTAGTTCGTCCAACACAGGTAGGGCTGAGGCATGATAAGATAAGTGACGGTTTGTGTAGCCTAGGTAATATATACTTAAGTGATGCAACAATATAAGTTCAGGTAGGGGAAATTCTTCTCGTACACATAGGAGTACATGGAATTTGGTAAGATTTCTTGATTTTTTTCTGCATTATACAAAGTTGCTGCATAGATTAAGTATAACTAAATAAGTGTCTTTTTATAATTAGAGGCTAATGAGTCATAGAAGTTGAATTAGGAGATAATCATTTGTAGTTGTAACTAGAGGTTTCTATAGGAAATTGAGGAGTATCCCAATCGAAATATAAATGCCTAAAAATGTCTGGGCTAAGAATTACATGTGAACTTGATGTCTGGGCTAAGAATTACATGTGAACTTGAAATTATATTGATCGGGTACAGGTCGAATGAAATTTTCCATAATTTCTTTTACTACTGGATTTATTACTTAAAAGGGTATAAACTTATCAAAATAAGATTTCTTAGAAGGTATGCGTGCTTTTATTTAAAACTTTTGTTTATGTATAATCCTATCTAATGCATGTACATGAATTATTTTGAATTTATTTTTACTCAAAGTAATCGTTATTTTCCTGTGTCATCTTCCTTATTATTTTTGTATATTCTATTTGTTTAGCAAATATATGAGCATCTTGTTTGTTTCAATCTTAGTCTTACCTGAACATAGTTGATTTATACGAAAGCTTATACATATATGAGTTCTGACTTGCATTATCTGATTTAGGTGGTCTGGGACTCACTGGCACGGGAGGTTCTTCGTCAAGCTTCTGGTATTTTTCACAGTGCACTTTAACAAAGTTATATTTTTATTTGTTCAAGTGTTGCCATTAACTTTATCTGCAATATTAGGCATTTTAGTTCATAGCCTGACTTTTCATCCATAACTTATCAAGTTCTATAACATGTTGCTGCTTCTTTTTAGTACATTGACATGATTGTGAAACCACTTTATAGGTGCATTATTCTTAGATGGATTGTTTCAGTCTAGGTGTCTTTTAGCCCTTATGTTGCAGAGGTTTTCTTCTTTACTCGTACACTTGCAGTGAGAACATGATTATTCGCTGCAAGTACATGTGTGTTGCAGAGTGCTTTTGAACTGATGAGAAGTTTCCTATATAATTGTTGATATTCAGCTATATTGATTCCAACATGGATATGTGTTTGTAACTTTGTACTGCTCTTAGATTTACCGTTTCAATATGCTGAATACGGTTTTCCTTTTTGATGATTCAGATCCATCTGCAAGTGCTGATCTGGCTGTGGTTCTGATGCAAGAAGGATTGGCACACATTCTTCTTATTGGTAAAAGGTAAGCTTGACAATCTCATAGTCCTATGAATACAAGTTTTAAACAGATCTTGAGCATCTCCTTTTCTTGTATTTAAACGAGATGTAATAGTTTAAAAGTCGAATGATTATGTCAGAATTTCTATATGTTGAGGCTGAGTTAAGTGTTAACTAGATTGTATGGCATAATTTTACCTTATGTACTTGTTACTGTTGTCTGAACAGTGTGACTATCACCCGTTCTCGTATAGAGTCTTCTATACCGCGCAAGCATGGACCGGCTATTGCAGGTTATGATAAGGTGAGTCTCTTACTTCTTTTTGTTTCATCTTTTGTATAATTTAATTATTTTGAACATGACGTCAAGTGAAATTGTGTTCTTAATTTTGATTTGCAGGCGTTAAATAAATTCTTTGACAATGTTCTACAGGTAGACTTTTGTCAACTTTCTTGATGTTGCTTAATTTCCAGAAGCAATATGTTATAGGTTCTTATTTCTGTTGCAGGCCTTTGTCAAGCATGTTGATTTCAAAGTAGTTCGCTGTGCTGTGATTGCAAGTCCAGGATTCACCAAGGTATTTTTTGTATAGTTACACTTCTTAGCTAGTCATACTTTTATGCTATGTTACAAGGGGTAGAACTTGCATATCTAATTATATCTGTACTATGCATATGTTAATTCAGTTGTGACATTTAAACTTTTTTGTGTTGAATGTGCAGGATCAGTTTCATCGTCACCTGTTGTTGGAAGCCGAGAGGAAGCAACTAAGACCTATAATAGAAAATAAGTCACGCATAATTCTTGTCCATACAACCTCGGGATACAAGTATGCCCTTCTTTCTCTCTCTCCTTGCCCTTCATCTGACATCTCAAAACGAGTCAATACATTTTTGTGCAAACCATATGATGTTAGACATGCGTGCTAGTCTAAAATTGACTAATATGTAGCAATACATTTTTGTGTACGCGATTCTCTCGTCAGTACTGCTATTTTAGTACAAATCGCCTTTATTATTTGTCTATTGTAGTTGATATGTAAAAGTTCAATTATTATCCAGCACGGGTGTTTAAGGCCGAGAGTTTTTTTTTTCCCTCGTTTTACAGACATAGTTTGAAAGAGGTTATGGATGCCCCAAATGTAATGACTATGATAAAAGATACAAAAGCTGCCAAAGAGGTACCTTCTGACCCTTGTCCAACTTGATATGATCTTTAATCTTTATTCTTGTGTTTGTTCAAGTCTTTTTTCGTATTTTTTGGGAATAACTGTTTTCCTTTCTCTTCCAATCTTAATCAGGTTCAAGCCCTAAAGGATTTTTTCAACATGCTTTCAAATGTTAGGTTCTATCCTTGGTCTCAATGTTCTATTTATATTTTTCTTAATAACTTTGGATGTTTCAGATTTTTATCTATCATATGCTAAGAGTAAACACAGATTGTTTGGTTTTGAATTTATTTGTTTGCTTTTTTTGGTGTTTATCGGTTATCTTGGGTTCTGTAAGAGGCCTGAGAGTTTCAAATAAAAGTTTGAGGTGTTTAACTATGCATGACGGATATCCCTGTTTAAGGAGAGGTGGACTAATCTGAAATATTTCTAGATTAAGAACAGATACGTTGTGTTATGATGGGCAGCTGGTGATATTGTTATCTCTGCGTGTGCCTTTTTTTAGGCCTTTCTACCAATTGAATGCTGAAAATTCATTTGCAACCAACCTTGGTTGAATTTATTTTCAGCAAGGATCCGTCATAATTCTCAGATATCCAATCATCAACAACTTTGAACTTCTATTAAATTTTAGGGCATCTCAGTTGTGTTCTTTCATCTTTTGATTTTATATGCAATTTTACTGTAGTAATAGGAGTATCTCTCTTCAATGCTTTGGCCATATCGGTACGAGAGAATTTATCATCTGATGTGCCCTCTCCTTTACTTTTTTGCAGACAGAAAATACGATAAGGAAGTCTTAATTAAAAATATGCGTGTGCTTGATTTTCTGGTTTTAAGGATATTTAGGTCTAAAAACTATAGTTACATTACATAATTTAGGGATGCTAGATGTAGAGGTCTTTTGCTAGAGCGAGTGCTTGTTTGAACCCCCCGCCCCGCCCCGCATATTTCATGAATTATGATATTAATTGGATATTTATGATACCGCTTTTCAAGCTTTCTGAAGAAACTAAGAAGGATAACATCTTTATTTAAAAACTTTTTTCCCTCTCTTGTTATAACTTCTTGCAATAAATGTAGTCATGTCCCTTTTTCTGGATCTCTTAACATTTATATTAATGAGCCCCTGCATGAAGTTTACTTCCAGTTGTCAACAAATAGATCCTTGTAGTGTGTTTCTTTACCCGAAACTTGTGAAAATTGAAGTTACTTTATTTGGACTTCTCTAGGATCCTGATCGTGCATGCTATGGACCAAAGCATGTTGAAGTTGCCCATGAGCGTCTGGCTATTCAGACACTTCTCATTACTGACGAGCTCTTTAGGTGAGTATCTTATGGTCCCAGGTTAATGGGGGCTTTATGAGCAATAAAATTAAACTGTATATAGCTTGATATAAATTTGCAACTCGTGGCATATTTCAAGTTCATAAGTATCTCTTTTTACGTGCTTAGCTTTTAAAATCGACATCTTTGGTTCATAGGATAATATGTAAACATGTATAACTATCTTCAAATCTCAACCAGTTATGTATGGGCCATTTCACTCGTGCTGTATATATATAGTTGGTTGATATCGTACATGAGAAATATCCAACTTCTTTTGATCTGTTGACAGGAGTTCTGATGTAGAAACGAGGAAAAAGTATGCTAATTTGGTCGATTCAGTCAAGGATTCAGGTGGTACTGCTCTCATTTTCTCGTCAATGCATGTCTCCGGAGAACGTGAGTATATACAATTCCTGTTAATTTCTTTTTCCTCCCAGCATTTTCATCTCTGCCTTTCCCTGTCGCCCCCTAGTAATGCTTAGAATGGTATTTTCCTTCTGTGCACATATTCATTGTCCCATAGTCGCTTCAAATCCTTTTTCCTTCCTACGAACACACGTTCTTTACTGCATATTTGTCAAGGCTGATTAAAACAACTTTTGGTTTTCGTCACAGAATTGAATCAGCTAACCGGCATTGCTGCAATCCTTCGTTTTCCTTTGCCGGAGCTGGAAGACATTGAGATGTGA

Although the embodiments of the present invention have been shown anddescribed, it is should be understood for those of ordinary skill in theart, various variation, modifications, substitutions, and improvementsmay be made to these embodiments without departing from the principleand spirit of the present invention, and the scope of the presentinvention is limited by the accompanying claims and their equivalents.

What is claimed is:
 1. A method for creating a germplasm of a malesterile crop by a gene editing, comprising: performing the gene editingon an exon region of a Ty-5 gene, and introducing a DNA sequencedeletion by using a repair mechanism of plants to double-strand breaks(DSBs), so as to reach a loss of function of the Ty-5 gene, therebyobtaining a male-sterile character.
 2. The method according to claim 1,wherein the gene editing is performed on the Ty-5 gene by using aCRISPR/Cas9 technology.
 3. The method according to claim 2, furthercomprising the following steps: (1) selection of gRNA target sitesselecting gRNA target sites in the exon region of the Ty-5 gene,according to a principle of the CRISPR/Cas9 technology, selecting a18-20 bp upstream of a protospacer-associated motif (PAM) as the gRNAtarget sites, wherein the gRNA target sites are 5′-N18-20NGG-3′, NGG isa PAM sequence, and N18-20 represents a recognition sequence of the18-20 bp; (2) designing oligo sequence primers based on the recognitionsequence as follows: Target-Sense: 5′-TTG-NNNNNNNNNNNNNNNNNNN, wherein Nrepresents a gRNAsense sequence; Target-Anti:5′-AAC-NNNNNNNNNNNNNNNNNNN, wherein N represents a reverse complement ofthe gRNAsense sequence; diluting the oligo sequence primers to aconcentration of 10 μM, taking 5 μL of each of the oligo sequenceprimers, and adding 15 μL water to the oligo sequence primers for mixinguniformly to obtain a first mixed solution, placing the first mixedsolution at 95° C. for 3 minutes, slowly cooling down the first mixedsolution to 25° C., and keeping the first mixed solution at 16° C. for 5minutes to complete a synthesis of an oligo dimer; (3) construction ofCRISPR/Cas9 recombinant vector and transformation of Agrobacteriumselecting a CRISPR/Cas9 kit VK005-14 from VIEWSOLID BIOTECH, Beijing,uniformly mixing 1 μL of the oligo dimer, 1 μL of a Cas9/gRNA vector, 1μL of Solution 1, 1 μL of Solution 2, and 6 μL of H₂O to obtain a secondmixed solution, reacting the second mixed solution at 16° C. for 2 h;transferring a ligated vector into E. coli competent state, picking asingle clone for a plasmid sequencing analysis, a sequencing primer issqprimer: 5′-GATGAAGTGGACGGAAGGAAGGAG-3′, as shown in SEQ ID NO: 5, andtransferring a positive plasmid to Agrobacterium GV3101; (4) croptransformation using a cotyledon of a crop as an explant, carrying out aplant regeneration by a leaf disc method, carrying out a positiveAgrobacterium GV3101-mediated transformation, performing a hygromycinresistance screening to obtain a regenerated plant.
 4. The methodaccording to claim 3, further comprising the following steps: (1)regenerated plant detection after gene editing: designing primersamplifying the gRNA target sites according to upstream and downstreamsequences of the RNA target site, extracting DNA of the regeneratedplant, using the DNA as a template for a PCR amplification to obtainamplified products, sequencing the amplified products, and performing asequencing analysis on the regenerated plant to determine whether thegene editing occurs and whether an editing type is a homozygousmutation; (2) after the sequencing analysis, identifying a pollencontent and germination of homozygous gene edited strains; wherein,results show that a homozygous edited Ty-5 plant created by the geneediting is a male sterile germplasm.
 5. A male sterile germplasmprepared by the method of claim
 1. 6. The male sterile germplasmaccording to claim 5, wherein the gene editing is performed on the Ty-5gene by using a CRISPR/Cas9 technology.
 7. The male sterile germlasmaccording to claim 5, wherein the method further comprises the followingsteps: (1) selection of gRNA target sites selecting gRNA target sites inthe exon region of the Ty-5 gene, according to a principle ofCRISPR/Cas9 target anchor, selecting a 18-20 bp upstream of aprotospacer-associated motif (PAM) as the gRNA target sites, wherein thegRNA target sites are 5′-N18-20NGG-3′, NGG is a PAM sequence, and N18-20represents a recognition sequence of the 18-20 bp; (2) designing oligosequence primers based on the recognition sequence as follows:Target-Sense: 5′-TTG-NNNNNNNNNNNNNNNNNNN, wherein N represents agRNAsense sequence; Target-Anti: 5′-AAC-NNNNNNNNNNNNNNNNNNN, wherein Nrepresents a reverse complement of the gRNAsense sequence; diluting theoligo sequence primers to a concentration of 10 μM, taking 5 μL of eachof the oligo sequence primers, and adding 15 μL water to the oligosequence primers for mixing uniformly to obtain a first mixed solution,placing the first mixed solution at 95° C. for 3 minutes, slowly coolingdown the first mixed solution to 25° C., and keeping the first mixedsolution at 16° C. for 5 minutes to complete a synthesis of an oligodimer; (3) construction of CRISPR/Cas9 recombinant vector andtransformation of Agrobacterium selecting a CRISPR/Cas9 kit VK005-14from VIEWSOLID BIOTECH, Beijing, uniformly mixing 1 μL of the oligodimer, 1 μL of a Cas9/gRNA vector, 1 μL of Solution 1, 1 μL of Solution2, and 6 μL of H₂O to obtain a second mixed solution, reacting thesecond mixed solution at 16° C. for 2 h; transferring a ligated vectorinto E. coli competent state, picking a single clone for a plasmidsequencing analysis, a sequencing primer is sqprimer:5′-GATGAAGTGGACGGAAGGAAGGAG-3′, as shown in SEQ ID NO: 5, andtransferring a positive plasmid to Agrobacterium GV3101; (4) croptransformation using a cotyledon of a crop as an explant, carrying out aplant regeneration by a leaf disc method, carrying out a positiveAgrobacterium GV3101-mediated transformation, performing a hygromycinresistance screening to obtain a regenerated plant.
 8. The male sterilegermlasm according to claim 5, wherein the method further comprises thefollowing steps: (1) regenerated plant detection after gene editing:designing primers amplifying the gRNA target sites according to upstreamand downstream sequences of the gRNA target site, extracting DNA of theregenerated plant, using the DNA as a template for a PCR amplificationto obtain amplified products, sequencing the amplified products, andperforming a sequencing analysis on the regenerated plant to determinewhether the gene editing occurs and whether an editing type is ahomozygous mutation; (2) after the sequencing analysis, identifying apollen content and germination of homozygous gene edited strains;wherein, results show that a homozygous edited Ty-5 plant created by thegene editing is a male sterile germplasm.